This invention is related to methods for inactivating micro-organisms using high pressure processing. The methods of the present invention may be applied preferably to food, cosmetic or pharmaceutical products.
Presently, the most conventional and widely used technique for inactivating micro-organisms and for sterilising products is thermal processing. Thermal sterilisation methods, such as the autoclaving of canned foods, are commonly used to extend the shelf life of food products. In the medical field, thermal processing is employed in sterilising, for example, various medical instruments. Although conventional thermal sterilisation methods are quite effective for inactivating a wide range of both vegetative and non-vegetative forms of micro-organisms on many types of products, there are several disadvantages. For example, thermal sterilisation often affects the natural taste, colour and/or texture of many products. Further, there are products which cannot be sterilised using conventional thermal processing because the structure would be destroyed. Thus, there is a need for effective alternative methods for inactivating micro-organisms that involve limited heat treatment and moreover do not affect many of the desired substances in products.
As an alternative to conventional thermal sterilisation techniques, the effectiveness of high pressure processing at ambient temperatures for inactivating micro-organisms in food has been known since the early 1900s. Although the inactivation of micro-organisms at high pressures is not completely understood, it is generally believed that micro-organisms are destroyed through altered permeability of the cell membranes from mechanical disruptions or through protein denaturation due to the disruption of hydrophobic and ionic bonds, and subsequent unfolding of the protein source. High pressure processing has demonstrated several advantages over conventional thermal sterilisation methods. Heat treatment of the product can be avoided or limited. The natural taste, colour and texture of many products are preserved, as well as the nutritional value and vitamin content. Further, chlorophyll and most aroma substances remain intact after high pressure processing. Physical damage to the food is also less using high pressure processing,
Japan introduced the first commercial food products to be sterilised using high pressure processing in 1990. Since then the high pressure sterilisation of foods has become widely accepted, and presently, there are several high pressure processed products on the market, including fruit, yoghurt, jam, jellies and fruit sauces.
Commercialised high pressure processing methods in the food industry generally involve placing food packed in a container inside a pressure vessel which contains a pressure transmitting medium (e.g. water). After the vessel is closed, the pressure is raised to a desired level by pumping the transmitting medium into the vessel by means of an external pressure intensifier. The vessel temperature can be maintained usually between +10 and +70xc2x0 C. The pressure is kept for a required time period. Then, the pressure is decreased. High pressure processing methods tend to vary depending on the settings for the pressure, temperature, time period and transmitting medium. Since the temperature of the vessel may be set, high pressure processing may also involve a combined application of elevated temperatures and high pressures.
In general, it has been found that pressure levels of approx. 600 MPa at 25xc2x0 C. are sufficient to totally inactivate vegetative forms of micro-organisms. Low pressure (400-600 MPa) applied for a restricted time period (5-10 min at 25xc2x0 C.) has proved effective in the destruction of common contaminating micro-organisms in food. In the case of pathogenic vegetative forms of micro-organisms, total inactivation required slightly higher levels of processing (500-600 MPa). To destroy yeast and mould, lower levels of pressure/time period were sufficient at 25xc2x0 C. Thus, high pressure processing at ambient temperatures has been proven to be very effective for inactivating non-spore forming pathogens, vegetative bacteria, yeast and moulds.
However, the effects of high pressures at ambient temperatures on the destruction of non-vegetative forms of micro-organisms, e.g. bacterial spores, has proven to be limited. Some research in the field indicates that the inactivation of such micro-organisms is possible using a combination of high pressures and elevated temperatures. For example, the inactivation of clostridia spores, bacilli and heat resistant moulds in technologically short time periods proved to be possible with processing at 900 MPa and simultaneously conditioning between 50 and 80xc2x0 C.
U.S. Pat. No. 6,086,936 describes a method for sterilising foods using both ultra-high pressures and high temperatures. The method involves heating a food to a pre-pressurised temperature, subjecting the food to ultra-high pressure, which instantaneously raises the temperature of the food, and then releasing the pressure so that the temperature returns to the original pre-pressurised temperature. The method leverages the adiabatic temperature rise which occurs when the food is hydrostatically pressurised, coupled with the lethality of the pressure, to achieve appropriate sterilisation conditions. The method disclosed however seems to be restricted to food, in particular non-dairy food products having a pH equal to or greater than 4.6.
In the cosmetic and pharmaceutical industries, there is a need for effective sterilisation techniques which do not reduce or alter the potency of the active substances in the product. Due to the high temperatures employed, it is not possible to use conventional heat sterilisation methods on many temperature-sensitive pharmaceutical products without negatively affecting the efficacy of the active substance. Therefore, a more gentle and equally effective means for sterilising such products would represent a significant technical advantage in the field. The potential to process cosmetic and pharmaceutical products using high pressure processing is already recognised as a viable option to current methods. However, an optimal method for inactivating micro-organisms in cosmetic and pharmaceutical products has not been disclosed by the prior art.
In xe2x80x9cPressure inactivation of micro-organisms at moderate temperaturesxe2x80x9d (Butz, P and Ludwig, H; Physica 139and140 B, 1986), xe2x80x9cHigh Pressure inactivation of bacteria and bacteria sporesxe2x80x9d (Butz, P, Ludwig, H et al; Pharm. Ind. 52, 1990), xe2x80x9cPressure induced germination and inactivation of Bacillus subtillis sporesxe2x80x9d (Sojka, B and Ludwig, H; Pharm. Ind. 56, 1994) and xe2x80x9cPressure and temperature induced inactivation of micro-organismsxe2x80x9d (Ludwig, H, Scigalla,W and Sojka, B; High Pressure Effects in Molecular Biophysics and Enzymology-Northrop and Royer, Chapter 22, 1996), methods using a combination of high pressures and elevated temperatures are discussed for inactivating micro-organisms, primarily bacteria and bacterial spores, with applications to the drug industry. Various parameters are investigated in order to ascertain the optimal conditions for destroying the micro-organisms mentioned. For example, in the article from Sojka and Ludwig in Pharm. Ind of 1994, the pressure inactivation of bacterial spores was studied in the range from 600 to 6000 bar at temperatures of 40 and 50xc2x0 C. At 40xc2x0 C., the number of germs could be reduced by a factor of 106 after 210 min. of pre-treatment at 600 bar and following inactivation at 5000 bar for some minutes. Raising the temperature to 50xc2x0 C., an alternating pressurisation at 600 and 5000 bar in intervals of 30 min. led to the complete inactivation of spores after an overall time of 180 min. In the aforementioned xe2x80x9cPressure and temperature induced inactivation of micro-organismsxe2x80x9d of 1996, results showed that pressure cycles are useful for the inactivating spores and further that the addition of ethanol promotes inactivation.
In the aforementioned references, several options are considered for yielding a method for destroying specific bacteria and bacterial spores whilst at the same time minimising the parameters of temperature, pressure and duration of the pressure applied. However, optimal parameters for a method of inactivating a wide range of micro-organisms, including non-vegetative forms, using high pressure processing that may also be safely applied to pharmaceutical products is yet to be determined.
It is therefore an object of the present invention to provide a more effective method for inactivating micro-organisms using high pressure processing.
The object of the present invention is achieved with the features of the claims.
The subject-matter according to the present invention has the particular advantage that it can be safely applied to pharmaceutical and cosmetic products without affecting the efficacy of the active substances in the products.
Another advantage of the present invention exists in providing an innovative method for inactivating micro-organisms using high pressure processing that is effective for inactivating even difficult-to-destroy non-vegetative bacteria and bacterial spores and at the same time, requires lower temperatures, lower pressures and shorter pressure application time periods as that of known methods.
The present invention provides an improved method for inactivating micro-organisms using high pressure processing. A combination of high pressures and elevated temperatures are used to inactivate even worst case bacterial spores. Although heat is applied to the product, the heat applied is generally significantly lower than that of conventional thermal sterilisation processes. Thus, the present method would still be considered as more desirable than conventional thermal processes for heat-sensitive products.
Further, the methods of the present invention introduce new factors which can be used to minimise the high pressure processing parameters necessary for inactivating specific micro-organisms, such as the required temperature and pressure applied to the product. It has been found that the effects of high pressure processing on the inactivation of micro-organisms may be greatly enhanced by pre-treating the product with specified amounts of oxygen. Since oxygen pre-treatment can promote inactivation, the processing parameters required in order to achieve the same micro-organism reduction criteria can be somewhat lower than the parameters required in methods without pre-treatment. In other words, lower processing temperatures and pressures at shorter time periods can be employed.
It was recognised surprisingly by experimentation that, when using products packed in flexible containers, oxygen pathways resulting from the high pressures of the transmitting medium applied appear to influence the amount of micro-organisms reduced. The presence of oxygen pathways contributed to increasing the overall effectiveness of the inactivation process. Thus, it is believed that by pre-treating the product using specified amounts of oxygen, one can further increase the effectiveness of the high pressure processing methods thereby simultaneously allowing a reduction of the processing parameters.
It is well known that oxygen is highly toxic because oxidation reactions can inactivate enzymes. An improved method which incorporates this factor can provide a very effective way to inactivate micro-organisms requiring minimised processing parameters, i.e. lower temperatures and pressures at shorter time intervals as that of known methods. Methods requiring minimised processing parameters are more desirable when processing sensitive products such as pharmaceuticals.
Another surprising factor is the influence of the various types of packaging on the high pressure processing methods. Several containers were tested and not all conventional containers were proven to be suitable under particular processing conditions. Thus, a consideration of the appropriate packaging has to be made in order to assure minimal damage to the product.
One preferred embodiment of the present invention is a method for inactivating micro-organisms using high pressure processing comprising the steps of; a) heating a product to a pre-pressurised temperature, b) placing the product in a pressure vessel, c) subjecting the product to a pressure at a pressurised temperature for a time period in the presence of a transmitting pressure fluid, d) reducing the pressure in the vessel after said time period, e) removing the product from said pressure vessel, wherein said method steps c) and d) may be repeated at least once before step e) is performed, said time period and said pressure being adjustable between repetitions of steps c) and d).
The aforementioned embodiment may further comprise an additional step before step b) of subjecting the product to an predetermined amount of oxygen for a time interval.
The product is heated to a pre-pressurised temperature using preferably a water bath. The pre-pressured temperature is preferably greater than ambient temperature and less than 70xc2x0 C. The transmitting pressure fluid in the pressure vessel is preferably water.
The pressure in the pressure vessel is preferably between 500 and 8000 bar, and preferably able to be incrementally increased and decreased. The pressurised temperature is preferably between 20 and 100xc2x0 C.
During the high pressure process, the pressure has a uniform effect on the entire pre-packaged product. The flexibility of the product""s container enables it to compensate for external pressure through a reduction in volume. Thus, flexible containers are preferable.
Another preferred embodiment of the present invention is a method for inactivating micro-organisms using high pressure processing comprising the steps of; a) subjecting a product to a pre-determined amount of oxygen for a time interval, b) placing the product in a pressure vessel, c) subjecting the product to a pressure at a pressurised temperature for a time period in the presence of a transmitting pressure fluid, d) reducing the pressure in the vessel after said time period, e) removing the product from said pressure vessel, wherein said method steps c) and d) may be repeated at least once before step e) is performed, said time period and said pressure being adjustable between repetitions of steps c) and d).
The pressure in the pressure vessel is preferably between 500 and 8000 bar, and preferably able to be incrementally increased and decreased. The pressurised temperature is preferably between 20 and 100xc2x0 C. The transmitting pressure fluid in the pressure vessel is preferably water. The product is preferably pre-packed in a flexible container.
The present invention can be utilised to process a variety of products, especially food, cosmetic and pharmaceutical products.